This proposal aims at establishing the role of endothelial cell expression of synectin, a ubiquitously expressed scaffold protein, on embryonic development. Previous studies have shown that global deletion of synectin causes an arterial-specific defect in vascular formation in adult mice. The first aim of this proposal will examine arterial development in synectin null embryos. The second part of Aim I will employ a cre/lox strategy to establish whether endothelial-specific inactivation of synectin expression is sufficient to account for the observed defects in arterial vascular formation in synectin-/- mice during development. Research proposed under Aim II will establish whether endothelial expression of synectin is sufficient to restore defective arterial development in synectin null embryos. This will be accomplished through the use of a double transgenic "Tet-OFF" system to specifically reintroduce synectin expression in endothelial cells of synectin null embryos. Taken together, these aims will establish the role of endothelial expression of synectin in arterial morphogenesis during mouse development. To implement this research plan, a variety of experimental techniques will be employed to quantitate overall vascular tree size and arterial vessel development and branching among the different genotypes. Whole mount immunostaining will be conducted using antibodies for CD31 and for arterial specific markers such as ephrin-B2 to identity gross abnormalities in vascular development. Micro computed tomography (micro-CT) will be used as a more quantitative measure of vascular development. In addition, embryonic development will be studied in a more controlled in vitro environment through the use of embryoid bodies. These embryoid bodies will be formed from stem cells derived from the different genotypes and analyzed using confocal microscopy and fractal analysis. [unreadable] [unreadable] [unreadable] [unreadable]